Inhibition stat-1

ABSTRACT

The present invention relates to inhibitors of the transcription factor STAT-1, their use as therapeutic means as well as their use for the prevention or therapy of cardio-vascular complications like restenosis after percutaneous angioplasty or stenosis of venous bypasses, the graft versus host reaction, the ischemia/refusion-related damage in the context of surgical interventions and organ transplantation respectively, immunological hypersensitivity reactions, in particular the allergic rhinitis, the drug and food allergies, in particular urticaria and celiac disease (sprue), contact eczema and the immune complex diseases, in particular alveolitis, arthritis, glomerulonephritis and allergic vasculitis, inflammatory chondro- and osteopathies, in particular arthrosis, gout, ostitis and osteomyelitis, polyneuritis as well as acute and subacute respectively, infection contingent and in particular post-infectious inflammatory diseases, in particular bronchitis, endocarditis, hepatitis, myocarditis, nephritis, pericarditis, peritonitis and pancreatitis, including the septic shock.

The present invention relates to the use of inhibitors of thetranscription factor STAT-1 for the manufacture of a medicament for theprevention or therapy of cardio-vascular complications like restenosisafter percutaneous angioplasty or stenosis of venous bypasses, the graftversus host reaction, the ischemia/refusion-related damage in thecontext of surgical interventions and organ transplantationrespectively, immunological hypersensitivity reactions, in particularthe allergic rhinitis, the drug and food allergies, in particularurticaria and celiac disease (sprue), contact eczema and the immunecomplex diseases, in particular alveolitis, arthritis,glomerulonephritis and allergic vasculitis, inflammatory chondro- andosteopathies, in particular arthrosis, gout, ostitis and osteomyelitis,polyneuritis as well as acute and subacute respectively, infectioncontingent and in particular post-infectious inflammatory diseases, inparticular bronchitis, endocarditis, hepatitis, myocarditis, nephritis,pericarditis, peritonitis and pancreatitis, including the septic shock.

It is a major aim of the decipherment of the human genome to identifymorbid genes (due to the mode of action of their products) and morbidchanges in the structure of these genes (polymorphisms) respectively andto assign them to a disease pattern. Therefore a causally determinedtherapy for most diseases has come into reach if it is accepted thatthese are caused by a defined number of gene products being expressedtoo strongly, too weakly or deficiently. In fact the usually singulargenetic defect (monogenetic diseases) is already known for a set ofhereditary diseases (e.g. cystic fibrosis) whereas the situation forother diseases (e.g. hypertension) turns out to be considerably morecomplex. The latter are obviously not the result of a single butmultiple genetic defects (polygenetic disease) predetermining theaffected persons to develop the disease in coincidence of certainenvironmental factors. Albeit this constraint the targeted interventionin the expression of one or multiple genes affords the opportunity of acause- and not only a symptom-based therapy.

Transcription factors are DNA-binding proteins that attach to thepromoter region of one or multiple genes inside the cell nucleus therebyregulating their expression, i.e. the regeneration of the proteins thesegenes are coding for. Besides the physiologically important role ofcontrolling developmental and differentiation processes in the humanbody, transcription factors display a high potential for eliciting adisease particularly if they activate the gene expression at a wrongpoint of time. In addition (possibly the same) transcription factors canblock genes with a protective function und act predisposing for theformation of a disease. Insofar the in the following described principleof an anti-transcription factor therapy aims at the inhibition of morbidgenes and the activation of protective genes in contrast.

Inflammation is a defence reaction of the organism and its tissuesagainst damaging stimuli aiming at the remediation of the damage or atleast its local limitation and at abolishing the cause of damage (e.g.invaded bacteria or foreign substances). The elicitors of aninflammation can be micro-organisms (bacteria, viruses, fungi orparasites), foreign substances (pollen, crystals of asbestos orsilicates), destruction of the tissue by mechanical impairment, chemicalnoxa and physical influences as well as elicitors from the body itself(collapsing tumour cells, extravasal blood, autoimmune reactions) orcrystals of intra-bodily precipitated substances (uric acid, calciumoxalate and calcium phosphate, cholesterol).

The rapid activation of mastocytes (inside the tissue) or of basophilegranulocytes in the blood is an example for the tripping of a verystrong acute-inflammatory response and is discriminatory forimmunological hypersensitivity reactions of the immediate type (humoralallergy type I). If the organism got into contact with an antigen (or anallergen, respectively, in the case of hypersensitivity) alreadybeforehand B-lymphocytes had been sensitised as a reaction to this. TheB-lymphocytes transform into plasmocytes in cooperation with previouslysensitised CD4-positive type 2 T-helper cells (Th2 cells) and startproducing antibodies of the IgE-type against the antigen. During thisdifferentiation process the co-stimulation of the B-lymphocytes via theCD40-receptor by the Th2-cells expressing the respective ligand (CD154)is of crucial importance. When the antigen-loaded IgE-antibodies bind tothe respective receptors (type Fc_(ε)) on the mastocytes these start torelease different mediators of inflammation especially histamine,interleukin-8, leukotrienes and tumour necrosis factor-α (TNFα).Consequence of which is the attraction of professional inflammatorycells especially of eosinophile and neutrophile granulocytes andmonocytes but also of T-lymphocytes on-the-spot (chemotaxis). At thesame time a histamine dependent vasodilatation and increase ofpermeability of the endothelial cells coating the interior vascular walltakes place. Due to the vascular dilatation the flow velocity decreasesfacilitating the establishment of the physical contact between theattracted leukocytes and the endothelial cells. These endothelial cellsbeing exposed to cytokines (e.g. TNFα) and thereby already activateddisplay an intensified expression of selectins on their luminal surface(e.g. E-selectin) causing a rolling along the endothelial cells of theleukocytes and thereby the activation of further adhesion molecules(integrins; e.g. intercellular adhesion molecule-1 [ICAM-1] or vascularcell adhesion molecule-1 [VCAM-1]). The leukocytes can now adhere to thevascular wall (margination) and the histamine-related increase inpermeability (loosening of the union of endothelial cells) favours theirmigration into the extravasal space (diapedese). At the same timeaugmented amounts of protein rich fluid (inflammatory exudate) attainthe interstitial space forming an oedema. Circumjacent nerve endings areirritated by the increasing pressure in the tissue and by furthermediators generated by the inflammatory cells and trigger pains makingthe damage of the tissue aware.

The granulocytes which have migrated to the site of inflammation and themonocytes which have re-differentiated into macrophages attempt toeliminate the causers of the inflammation by phagocytosis and lysisrespectively thereby triggering the release of inter alia proteolyticenzymes and oxygen radicals that may damage also the surrounding tissue.In particular the activation of the macrophages can account in many waysfor the fact (e.g. by the release of further cytokines likeinterleukin-1β or interleukin-6) that the entire organism is involved bythe primarily local inflammatory response in terms of an acute phaseresponse. Representative characteristics of an acute phase response arefatigue, lassitude and fever, an increased release of leukocytes fromthe bone marrow (leukocytosis), the detection of acute phase proteins inthe blood (e.g. C-reactive protein), the stimulation of the immunesystem as well as weight loss due to a changed status of the metabolism.

If the cause of the inflammation can be eliminated the process of woundhealing falls into line with the destroyed tissue being repaired. Atbest this amounts to an entire re-establishment (restitutio adintegrum), whereas bigger lesions or an excessive production ofconnective tissue (especially collagen) result in the formation of ascar which is possibly associated with considerable dysfunctionsdepending on the affected tissue. If the cause cannot be eliminated atonce (foreign substances or wound infection) the wound healing isdelayed at simultaneous increase of the immigration and activity of thephagocytes bringing about the doom of the tissue (necrosis) up to theformation of cavities (abscess). The result is almost always a scarredre-structuring of the tissue with a respective loss of function. If thelocal limitation of the inflammation which is derived from the causativeagent does not succeed, the inflammation spreads over the entireorganism via the lymphatic system. The consequence is a sepsis with apossibly fatal upshot (septic shock).

Wound healing is also interfered with if the inflammatory and thehealing process are in balance. The result is a chronic inflammationwhich may be fibrosing (excessive synthesis of collagen) orgranulomatous (organisation of inflammatory cells into a granulationtissue) and usually brings about a continuous destruction and increasingconstraint of functionality of the affected tissue respectively.

Besides the depicted common inflammatory response which may degeneratechronically there are inflammatory diseases that exhibit both commongrounds and distinct differences with regard to the underlyingpathogenesis. Two inflammatory diseases of such kind are for examplecomplications after cardio-surgical interventions and the immunologicalhypersensitivity reactions which more space in this specification isdedicated to because of their enormous clinical relevance.

The balloon-tipped catheter based mechanical dilatation (percutaneousangioplasty) and the bypassing of arteriosclerotically stenosed arteriesby means of venous bypasses respectively still constitute the therapiesof choice in patients with coronary and peripheral circulatory disordersrespectively in order to provide protection against an imminentinfarction or organ failure. But the rate of re-occlusion (restenosis)of the arteries which were mechanically dilated and (in the majority ofcases) treated with a metallic vascular support (stent) appearsunacceptably high with 20-50% within 6 months. Also the rate ofre-occlusion of aortocoronary and peripheral venous bypassesrespectively with 50-70% after 5 years is more than dissatisfactory forthe treated patients in particular against the background of the riskaround the procedure and the postoperative risk respectively. Presumablybecause of the damage of the vascular wall (hereby both the endothelialand the smooth muscle cells being affected) the restenosis afterangioplasty shows particularly in the early stage a pronouncedinflammatory component being characterised inter alia by theinfiltration of the vascular wall with professional inflammatory cells(above all monocytes and T-lymphocytes). The fibro-proliferatingstenosis formation (intimal hyperplasia) in aortocoronary and peripheralvenous bypasses respectively seems to be based also on a inflammatoryreaction which in particular is caused by mechanical and physical noxa.It has been known for a long time also that the so calledischemia/refusion-related damage in the context of surgicalinterventions or organ transplantations is accompanied by aninflammatory-based tissue damage in which the interaction betweenendothelial cells and professional inflammatory cells (above allgranulocytes but also monocytes and T-cells) as well as the release oftissue damaging substances (oxygen radicals, cytokines) play a quitecrucial role.

In connection with the mentioned cardio-vascular complications it isimportant that there are protective mechanisms, above all in theendothelial and smooth muscle cells of the vascular wall, which help tolimit the extent of the inflammatory response and the subsequentadaptive re-structuring of the tissue. To this for example belongs thesynthesis of nitric oxide (NO) by the NO-synthase in the endothelialcells. NO, probably featuring the endogenous antagonist of the oxygenradical superoxide, inhibits inter alia the expression ofpro-inflammatory chemokines (e.g. monocyte chemoattractant protein-1,MCP-1) and of adhesion molecules (e.g. ICAM-1) in endothelial cells, theexpression of receptors for growth factors in smooth muscle cells (e.g.endothelin B-receptor) as well as the release of growth factors fromleukocytes. Insofar it is easy to comprehend that a mechanical damagejust as a functional damage of the endothelium (e.g. by acytokine-induced reduction of the expression of the NO-synthase in thesecells) counteract the processes of inflammation and subsequentfibro-proliferating re-structuring of the vascular wall which form thebasis for the mentioned cardio-vascular complications.

All previous attempts to check the restenosis after angioplastymedicamentously have not achieved the desired effect in the majority ofpatients. At present two local principles of therapy are favoured: thealready approved vascular brachytherapy, a method for checking the cellgrowth by short-time radioactive irradiation of the dilated vascularsection and the drug-eluting stents which are still in the clinicaltrial. This method comprises polymer coated stents which are“impregnated” by growth inhibiting medicaments (cytostatic andimmunosuppressive agents) and release them slowly during a period ofseveral weeks. Most recent clinical studies prove that both therapeuticapproaches are not exempt from to some extent serious problems (e.g.in-stent-thrombosis running the danger of an infarction) despite ofencouraging results at the beginning.

Besides the already delineated immunological type I-incompatibilityreaction there are in principle four other forms of allergy anddysfunctions in the immune regulation respectively. The type I-reactionitself can in principal be sorted into two phases after allergisationwas accomplished: the rapid release and regeneration of vascularlyactive inflammatory mediators from IgE-spiked mastocytes and the latereaction which is mediated by the attracted eosinophile and neutrophilegranulocytes. The complete type I-reaction can take place either locallyor systemically in dependence on the exposure to the allergen. Allergensin the respiratory air elicit reactions in the respiratory tract,typically accompanied by mucosal oedemas and hypersecretion (allergicrhinopathy, hay fever) as well as bronchospasm (asthma) whereasallergens in the nourishment elicit gastrointestinal symptoms likenausea, vomitus and diarrhoea. The skin reacts on allergens with itchingand urticaria as well as atopic dermatitis (neurodermatitis) But if theallergen gains direct access to the bloodstream (e.g. infusion of bloodproducts, medicaments) or if the exposure to the allergen is especiallystrong, a systemic immediate reaction results possibly entailing alife-threatening decrease of the blood pressure (anaphylactic shock).

In the case of the type II-reaction antigenically active cells (e.g.extraneous blood cells) or extracellular proteins (e.g.medicament-induced changes at the surface of a cell naturally producedin the body) take centre stage. After allergisation the second contactleads to the production of allergen-specific antibodies of the IgG- andIgM-type which bind to the allergenic cell in great quantities(opsonisation). Hereby the complement system (formation of a membraneattacking complex) and a special subpopulation of lymphocytes, thenatural killer cells (NK-cells), are activated. The result is adestruction of the allergenic cell by cytolysis. A similar reaction iselicited when auto-antibodies attach to structures that are naturallyproduced in the body such as the basal membrane of the glomerularcapillaries and thereby eliciting a rapidly progressiveglomerulonephritis with imminent renal insufficiency. Besides the type 1T-helper cells (Th1-cells, see below) the activated NK-cells are themain producers of interferon-γ, a cytokine that massively intensifiesthe inflammatory response in particular by the activation ofmacrophages.

The type III-reaction is characterised by the formation and depositionof immune-complexes (antigen-antibody-complexes) with subsequentactivation of the complement system and phagocytes (granulocytes,macrophages). They circulate in the blood and successively depositmainly in the capillaries of the renal glomeruli but also in the jointsor in the skin. The hereby elicited inflammatory response may bringabout a (immune-complex-) glomerulonephritis, pains in the joints aswell as urticaria. Infections can also elicit a systemic typeIII-reaction if the immune system fails to eliminate the causative agent(e.g. streptococci). Representative local type III-reactions are the socalled Arthus-reaction in the skin after an immunisation or theexogenous allergic alveolitis in the case of deposition ofantigen-antibody-complexes in the lung (e.g. bird-breeder's lung). Thesystemic lupus erythematodes is a type III-reaction as well but in termsof an autoimmune disease due to the formation of auto-antibodies.

In contrast to the hypersensitivity reactions mentioned before the typeIV-reaction is not humoral but cell constrained and reaches its maximumusually not until after several days (delayed type of reaction ordelayed type hypersensitivity). Elicitors are mainly proteins, invadedforeign organisms (bacteria, viruses, fungi and parasites), otherforeign proteins (e.g. wheat-derived gliadin in the case of celiacdisease) as well as haptens (medicaments, metals [e.g. nickel in thecase of contact dermatitis], cosmetics and plant components). Theprimary rejection of transplanted organs is also a type IV-reaction. Theantigen is phygocytised by (tissue) macrophages, processed and presentedto naive T-helper cells (CD4-positive); the allergisation of theT-helper cells takes several days. At the second contact the in such away sensitised T-helper cells alter in Th1-cells; thereby theCD154-mediated co-stimulation of the antigen-presenting cell (this oneexpresses the CD40-receptor) plays an important role because thissignalling pathway triggers the release of interleukin-12 from themacrophages. Interleukin-12 initiates the differentiation andproliferation of the T-helper cells. The Th1-cells on their part excitethe formation of monocytes in the bone marrow by certain growth factors(e.g. GM-CSF), recruit these by means of certain chemokines (e.g. MIF)and activate them by the release of IFNγ. The hence resulting verystrong inflammatory response may destroy tissue normally produced in thebody (e.g. tuberculosis) or transplanted tissue in a large scale.Moreover CD8-positive cytotoxic T-cells are involved in the transplantrejection (cytolysis) with the CD8-positive cytotoxic T-cells being ableto recognise their target (the foreign cell surface) and to “arm”themselves accordingly only by a preceding antigen-presentation like theCD4-positive Th1-cells.

A dysfunction of the immune regulation similar to a type IV-reactionforms the basis for e.g. the rheumatoid arthritis or the multiplesclerosis (auto-reactive Th1-cells) as well as for diabetes mellitus(auto-reactive cytotoxic T-cells). T-cells being directed againstcertain antigens of the causative agent (e.g. streptococci) whichcross-react with auto-antigens (produced in the body; molecular mimicry)might potentially play a role at these autoimmune diseases besidesbacterial super-antigens (e.g. the causative agent of TBC) and theaccording genetic predisposition (MHC-proteins, Th1/Th2-imbalance). Incontrast, type V-reactions may be evoked inter alia by activating orblocking auto-antibodies of hormone—(e.g. thyrotropin in the case ofBasedow's disease) or neurotransmitter-receptors (e.g. acetylcholine inthe case of myasthenia gravis).

Comparable with the transplant rejection—yet in the reverse sense—is thegraft versus host disease (GVHD) which appears in the course ofallogenic bone marrow transplantations (between genetically nonidentical individuals) in about 40% of the recipients. During theacute-phase lasting up to three months the T-cells of the donor whichhave been transfused with the stem cells attack the host organism. Theresulting possibly severe inflammation response becomes manifestpreferably in the skin, the gastrointestinal tract and in the liver.

For the treatment of acute inflammatory diseases in dependence on to theassumed cause usually non-steroidal antiphlogistics (NSAIDs, inter aliainhibition of the synthesis of prostaglandins) and/or anti-infectiousagents (devitalisation of bacteria, fungi or parasites) and antiviralchemotherapeutics respectively, contingently also glucocorticoids(general inhibitors of gene expression) in a local application, areutilised. In the case of severe or chronically recurring inflammatorydiseases glucocorticoids or immunosuppressive agents (inhibition of theT-cell-activation) or cytostatics such as methotrexate are systemicallyadministered. This also applies to the transplantation of organs andbone marrow respectively. Despite of their undisputable therapeuticeffect a systemic administration of the mentioned pharmaceuticals canevoke severe side effects especially when permanently used. So forexample up to 25% of the patients who take methotrexate for 2 or moreyears develop a severe cirrhosis of the liver. More recent active agentsthat are used in particular with chronically recurring inflammatorydiseases block the pro-inflammatory effect of TNFα: antibodies directedagainst the cytokine itself and its receptor respectively, low-molecularantagonists of the receptor as well as a recombinantly produced, solublereceptor protein that traps the cytokine. But there is a growing numberof indications for an increased incidence of infectious diseases duringthe therapy with the receptor protein (inter alia tuberculosis), andabout 40% of the patients do not seem to respond to the therapy at all(non-responder). Also for the approved humanised TNFα-antibody there areaccording warning notices concerning the incidence of infections rangingup to sepsis 2-4 years after the start of the therapy. Moreover bothactive agents are contraindicated during an acute incident. In additionlow-molecular antagonists of the receptor are approved for leukotrieneswhich are mainly used in the therapy of asthma as well as inhibitors ofthe cyclooxigenase-2, a new group of non-steroidal antiphlogistics(NSAIDs) with considerably reduced gastrointestinal side effects incomparison to the classical NSAIDs. Moreover there is a series offurther—usually humanised—antibodies or antisense-oligonucleotide basedapproaches against adhesion molecules of leukocytes and endothelialcells respectively, cytokine receptors of T-helper cells orIgE-antibodies which are residing in different phases of the clinicaltrial. To refrain from the glucocorticoids and the anti-infectiousagents as a group, the mentioned pharmaceuticals have in common to bedirected specifically against a target molecule which is of relevancefor the therapy.

The present invention is therefore based on the problem to providesubstances for the prevention or therapy of cardio-vascularcomplications like restenosis after percutaneous angioplasty or stenosisof venous bypasses, the graft versus host reaction, theischemia/refusion-related damage in the context of surgicalinterventions and organ transplantation respectively, immunologicalhypersensitivity reactions, in particular the allergic rhinitis, thedrug and food allergies, in particular urticaria and celiac disease(sprue), contact eczema and the immune complex diseases, in particularalveolitis, arthritis, glomerulonephritis and allergic vasculitis,inflammatory chondro- and osteopathies, in particular arthrosis, gout,ostitis and osteomyelitis, polyneuritis as well as acute and subacuterespectively, infection contingent and in particular post-infectiousinflammatory diseases, in particular bronchitis, endocarditis,hepatitis, myocarditis, nephritis, pericarditis, peritonitis andpancreatitis, including the septic shock which constitute a broader(knowingly not mono-specific) and thereby a potentially more effectivetherapeutic approach.

The problem is solved by the subject-matter defined by the patentclaims.

The invention is elucidated by the following figures in greater detail:

FIG. 1 shows the inhibition of the cytokine-stimulated expression ofCD40 (a, c, d and e), E-selectin and MCP-1 (a) and of theCD40-ligand-induction of the interleukin-12p40-expression (b) incultivated human endothelial cells by neutralisation of thetranscription factor STAT-1 by means of an according cis-element-decoy(SEQ ID NO: 33). (a) Representative RT-PCR-analysis of the E-selectin,MCP-1 and CD40 mRNA-expression (in addition the densitometric analysis(“intensity”) specified in % of the stimulated control and referring tothe internal standard EF-1) in endothelial cells which had beenpre-incubated with a STAT-1 (SEQ ID NO: 33) or NF-κB cis-element decoy(10 μM) for 4 hours and subsequently incubated with 100 U/ml tumournecrosis factor-α and 1000 U/ml interferon-γ for 9 hours. (b)Representative RT-PCR-analysis of the mRNA-expression ofinterleukin-12p40 (in addition the densitometric analysis (“intensity”)specified in % of the stimulated control and referring to the internalstandard rp132) in endothelial cells which had been pre-incubated with aSTAT-1 cis-element decoy (10 μM; SEQ ID NO: 33) for 4 hours andsubsequently incubated with about 670000 P3xTB.A7-cells/ml (these mousemyeloma cells stably express the human CD40-ligand CD154) and 1000 U/mlinterferon-γ for 12 hours. (c) Representative RT-PCR-analysis of theCD40 mRNA-expression (in addition the densitometric analysis(“intensity”) specified in % of the stimulated control and referring tothe internal standard EF-1) in endothelial cells which had beenpre-incubated with a STAT-1 cis-element decoy (SEQ ID NO: 33) or therespective control oligonucleotide (STAT-1-25mut) for 4 hours(concentration 10 μM) and subsequently incubated with 100 U/ml tumournecrosis factor-α and 1000 U/ml interferon-γ for 9 hours. (d)Statistical summary of 5 independent experiments on the effect of theSTAT-1 cis element decoys (SEQ ID NO: 33) on the cytokine-stimulatedCD40 mRNA-expression the cultivated endothelial cells (*p<0.05 versusthe stimulated control cells). (e) Representative western-blot-analysisin addition to the densitometric analysis (“intensity” specified in % ofthe stimulated control and referring to the internal standard β-actin)of the effect of the STAT-1 cis element decoys (SEQ ID NO: 33) on thecytokine-stimulated CD40 protein-expression the cultivated endothelialcells after 24 hours. Comparable results were obtained in furtherexperiments.

FIG. 2 shows the inhibition of the cytokine-induced expression of theCD40 gene in human cultivated endothelial cells by theantisense-oligonucleotide based down regulation of the expression of thetranscription factor STAT-1. (a) Expression of the CD40- andSTAT-1-protein respectively under resting conditions and afterincubation of the cells with 100 U/ml tumour necrosis factor-α and 1000U/ml interferon-γ for 14 hours. The left panel of the picture shows thestatistical summary of 2-4 experiments with different batches of cells,the right panel of the picture shows each a representativewestern-blot-analysis in addition to the densitometric analysis(“intensity”) specified in % of the non-stimulated control and referringto the internal standard β-actin (*p<0.05 versus the non-stimulatedcontrol cells). (b) Comparable inhibition of the CD40- andSTAT-1-protein expression in stimulated endothelial cells by apre-treatment with a STAT-1-antisense-oligonucleotide (1 μM; SEQ ID NO:33) for 24 hours. Summary of 2 experiments (left panel of the picture;*p<0.05 versus the stimulated control cells) and representativewestern-blot-analysis (right panel of the picture).

FIG. 3 shows the inhibition of the expression of the transcriptionfactor IRF-1 in the monocyte-cell-line THP-1 (a) as well as of theinducible isoform of the NO-synthase in cultivated human smoothmuscle-cells (b) by the neutralisation of the transcription factorSTAT-1 by means of a respective cis-element decoy (SEQ ID NO: 33). (a)Representative western-blot-analysis in addition to the densitometricanalysis (“intensity”) specified in % of the stimulated control andreferring to the internal standard 0-actin. The cultivated THP-1-cellswere pre-incubated with the cis-element decoy (10 μM) for 4 hours andsubsequently incubated with 100 U/ml tumour necrosis factor-α and 1000U/ml interferon-γ for 3 hours. (b) Left panel of the picture:statistical summary of 3 experiments with different batches ofcultivated human smooth muscle cells which had been pre-incubated with aSTAT-1 (SEQ ID NO: 33), a NF-κB or a GATA-2 cis-element decoy (10 μM)for 4 hours and subsequently incubated with 1000 U/ml interferon-γ, 60U/ml interleukin-1β, 100 U/ml tumour necrosis factor-α and 1 μg/ml of abacterial lipopolysaccharide for 9 hours. RT-PCR-analysis of themRNA-expression for the inducible isoform of the NO-synthase (*p<0.05versus the stimulated cells=100%). Right panel of the picture:statistical summary of 3 experiments with different batches of cells andrepresentative western-blot-analysis of the inhibition of thecytokine-stimulated expression of the NO-synthase protein (after 20hours of exposition) by pre-incubation with the STAT-1 (SEQ ID NO: 33)and NF-κB cis-element decoy respectively (*p<0.05 versus the stimulatedcells=100%).

FIG. 4 shows the neutralisation of endogenous STAT-1 in extracts of cellnuclei of the monocyte-cell-line THP1 by different cis-element decoys(SEQ ID NO: 17, 25, 29, 31, 33, 35, 37, 39 and the mutatedcontrol-oligonucleotides STAT-1-19mut and STAT-1-25mut). RepresentativeEMSA-analysis. Cultivated THP-1 cells were incubated with 100 U/mltumour necrosis factor-α and 1000 U/ml interferon-γ for 3 hours andsubsequently used for the preparation of nuclear extracts. The nuclearextract of the cells was co-incubated with the [³²P]-labelled doublestranded SIE-oligonucleotide (Santa Cruz Biotochnologie, Heidelberg,Germany) and the respective cis-element-decoys andcontrol-oligonucleotides respectively at room temperature for 20 minutesand was subsequently subjected to the electrophoretic mobilityshift-analysis.

FIG. 5 shows the effect of selected STAT-1 cis-element decoys (SEQ IDNO: 17, 31, 35, 37) on the expression of E-selectin and MCP-1 mRNA inhuman smooth muscle cells from the thymus vein. The cultivated cells(passage 2) were pre-incubated with the respective cis-element decoys(10 μM) for 4 hours and subsequently incubated with 100 U/ml tumournecrosis factor-α and 1000 U/ml interferon-γ for 9 hours. RepresentativeRT-PCR-analysis, comparable results were obtained in furtherexperiments.

FIG. 6 schematically shows the structure of theSTAT-1-antisense-expression vector pCI/Stat1 AS in terms of a gene map.

FIG. 7 shows the result of the neutralisation of STAT-1 in humancultivated endothelial cells by different cis-element decoys (SEQ ID NO:17, 19, 27, 33 and 39). Representative EMSA-analysis in addition to thedensitometric analysis (“intensity”). The cultivated endothelial cellswere incubated with the decoy-oligonucleotides (10 μmol/l) for 4 hoursand subsequently stimulated with 100 U/ml tumour necrosis factor-α and1000 U/ml interferon-γ for 30 min. For the EMSA-analysis nuclearextracts of the stimulated cells and the [³²P]-labelled double strandedSIE-oligonucleotide (Santa Cruz Biotechnologie, Heidelberg, Germany)were used.

FIG. 8 shows the histological analysis of the effect of a STAT-1decoy-oligonucleotide (STAT-icons, 10 nmol, SEQ ID NO: 19) but not of amutated control-oligonucleotide (STAT-1mut, 10 nmol, SEQ ID NO: 61) onthe DNCB-induced contact-dermatitis in male guinea pigs (original ×400,typical result of 17 examined guinea pigs in total).

The inventors have characterised the transcription factors which takepart in the cytokine-mediated increase of the expression ofpro-inflammatory gene products (CD40, E-selectin, inducible isoform ofthe NO-synthase, interleukin-12 [p40], MCP-1) in human endothelial- andsmooth muscle cells as well as in monocytes. Thereby it could be shownthat there is a synergism between the transcription factors nuclearfactor κB (NF-κB) and the signal transducer and activator oftranscription-1 (STAT-1) in the case of the stimulation of thecultivated endothelial cells with TNFα and CD154 respectively incombination with IFNγ. The same holds true for the cultivated smoothmuscle cells and monocytes respectively.

IFNγ alone was able to increase the expression of CD40 in humanendothelial cells but not the one of E-selectin or interleukin-12. Forthe expression of those two gene products which are hardly andnon-constitutively respectively expressed in endothelial cells asimultaneous stimulation of the cells with TNFα: (E-selectin) and CD154(interleukin-12) respectively is essential. Furthermore the de novoexpression of an additional transcription factor, the interferonregulatory factor-1 (IRF-1), is necessary for the IFNγ-mediated increaseof the expression of CD40 but not of E-selectin in the endothelial cellsand monocytes. In the scope of these analyses it could be shown that theIRF-1-protein expression is considerably weaker in the case of themono-stimulation of the cells with IFNγ and in particular with TNFα thanin the presence of both cytokines. According to this the transcriptionfactors NF-κKB and STAT-1 act synergistically in the case of thetranscription of the IRF-1 gene, too (Ohmori et al., J. Biol. Chem.,(1997), 272, 14899).

STAT-1 (GenBank Accession Number NM007315 and XM010893 andhttp://transfac.gbf.de/cgi-bin/qt/getEntry.p1?t0149 respectively)belongs to a group of transcription factors which comprises at least 6members. The product of the STAT-1 gene is expressed constitutively bymost of the cells but usually exists as an inactive monomeric protein(91 kDa) in the cytoplasm. The tyrosine-phosphorylation of thisp91-subunit and the subsequent association (dimerisation) of two of suchp91-subunits (called STAT-1α) enables the transport of the from now onactive transcription factor into the nucleus of the cell. Ahetero-dimerisation with the p84-subunit of STAT-1β (differentiallyspliced product of the same gene) is also possible. The phosphorylationof the constitutively existing subunits occurs via cytoplasmicjanus-kinases in dependency of the stimulus. So both janus-kinases (Jak1and Jak2) are stimulated by IFNα (recruited better to the interferonreceptor); on the contrary, the most important stimulus in terms of(patho)physiology for the activation of STAT-1, IFNγ, only stimulatesJak2. Different growth factors and peptide hormones (e.g. angiotensinII) activate STAT-1 as well; besides the intrinsic (growth factor)receptor-tyrosine-kinases also a mitogen-activated protein kinase(MAP-kinase) plays a role at this. In contrast to STAT-1α STAT-1β has notransactivating, i.e. the gene expression stimulating, activity.

STAT-1 takes part in the expression of a series of potentiallypro-inflammatory gene products in leukocytes, endothelial cells andsmooth muscle cells whereby the activation of the transcription factorusually occurs in an IFNγ-dependent way. An exception is in particularthe STAT-1-dependent expression of interleukin-6 in angiotensinII-stimulated smooth vascular muscle cells (Schieffer et al., Circ. Res.(2000), 87, 1195).

One aspect of the present invention relates to the use of inhibitors ofthe activity of the transcription factor STAT-1 for the manufacture of amedicament for the prevention or therapy of cardio-vascularcomplications like restenosis after percutaneous angioplasty or stenosisof venous bypasses, the graft versus host reaction, theischemia/refusion-related damage in the context of surgicalinterventions and organ transplantation respectively, immunologicalhypersensitivity reactions, in particular the allergic rhinitis, thedrug and food allergies, in particular urticaria and celiac disease(sprue), contact eczema and the immune complex diseases, in particularalveolitis, arthritis, glomerulonephritis and allergic vasculitis,inflammatory chondro- and osteopathies, in particular arthrosis, gout,ostitis and osteomyelitis, polyneuritis as well as acute and subacuterespectively, infection contingent and in particular post-infectiousinflammatory diseases, in particular bronchitis, endocarditis,hepatitis, myocarditis, nephritis, pericarditis, peritonitis andpancreatitis, including the septic shock, for the attenuation of theSTAT-1-dependent expression of pro-inflammatory gene products in thescope of inflammatory responses.

Proteins, including also STAT-1, can be inhibited in their activity invery different ways. So e.g. anti-STAT-1-antibodies as well as naturalor synthetic substances can be used which reduce the STAT-1-interactionwith the DNA, i.e. reducing the transactivation activity. Further thesignalling pathways (Jak1, Jak2, receptor tyrosine-kinases,MAP-kinases), which lead to the activation of STAT-1, could beinhibited. Preferred methods for the specific inhibition of the activityof STAT-1 are:

-   1. The neutralisation of the activated transcription factor by a    decoy-oligonucleotide,-   2. the inhibition of the STAT-1-protein expression by means of an    antisense-oligonucleotide,-   3. the inhibition of the STAT-1-protein expression by means of an    antisense-expression vector, and-   4. the inhibition of the STAT-1-protein expression by the    application of double stranded RNA-oligonucleotides    (dsRNA-interference).

The herein used terms “decoy-oligonucleotide” or “cis-element decoy”refer to a double stranded DNA-molecule and a double strandedDNA-oligonucleotide respectively. Both DNA-strands exhibit acomplementary sequence. In the present invention the cis-element decoyexhibits a sequence which is in accordance or similar to the naturalSTAT-1 core binding-sequence in the genome and which is bound by thetranscription factor STAT-1 inside the cell. Thus the cis-element decoyacts as a molecule for the competitive inhibition (betterneutralisation) of STAT-1.

A preferred method for the specific inhibition of the STAT-1-activity isthe use of double stranded DNA-oligonucleotides, also called cis-elementdecoy or decoy-oligonucleotide, containing a binding site for STAT-1.The exogenous supply of a great number of transcription factor bindingsites to a cell, in particular in a much higher number then present inthe genome, generates a situation in which the majority of a certaintranscription factor binds specifically to the respective cis-elementdecoy and not to its endogenous target binding site. This approach forthe inhibition of the binding of transcription factors to theirendogenous binding site is also called squelching. Squelching (or betterneutralisation) of transcription factors using cis-element decoys wasapplied successfully to inhibit the growth of cells. HerebyDNA-fragments were used which contained the specific transcriptionfactor binding site of the transcription factor E2F (Morishita et al.,PNAS (1995) 92, 5855).

The sequence of a nucleic acid which is used for the prevention of thebinding of the transcription factor STAT-1 is e.g. the sequence whichSTAT-1 naturally binds to inside the cell. STAT-1 binds specifically tothe motive with the sequence 5′-NNNSANTTCCGGGAANTGNSN-3′ in which thedenotation is as follows: N=A, T, C or G and S=C or G. The exactconsensus with the underlined bases and the distance between these basesare imperative for an effective binding of STAT-1. Therefore thecis-element decoy according to the invention may exhibit the following11-mer consensus-core binding sequence: 5′-NTTNCBGDAAN-3′ (SEQ ID NO: 1)in which the denotation is as follows: B=C, G or T, D=A, G or T and N=A,T, C or G. Furthermore the cis-element decoy can be larger than the11-mer core binding site and be elongated at the 5′-end and/or at the3′-end. According mutations in the region of the core binding sequencelead to the deprivation of the binding of STAT-1 to thedecoy-oligonucleotide.

Since the cis-element decoy is a double stranded nucleic acid theDNA-oligonucleotide according to the invention comprises not only thesense- or forward-sequence but also the complementary antisense- orreverse-sequence. Preferred DNA-oligonucleotides according to theinvention exhibit an 11-mer core binding sequence for STAT-1:

5′-ATTACCGGAAG-3′, (SEQ ID NO: 3) 5′-ATTCCGGTAAG-3′, (SEQ ID NO: 5)5′-ATTCCTGGAAG-3′, (SEQ ID NO: 7) 5′-ATTCCTGTAAG-3′, (SEQ ID NO: 9)5′-GTTCCAGGAAC-3′, (SEQ ID NO: 11) 5′-GTTCCCGGAAG-3′, (SEQ ID NO: 13)5′-GTTCCGGGAAC-3′, (SEQ ID NO: 15)whereas the respective complementary sequences are not depicted here.But the cis-element decoy can also exhibit a sequence differing from theprevious sequence and be longer than an 11-mer.

Particularly preferred are the following sequences:

(SEQ ID NO: 17): 5′-TGTGAATTACCGGAAGTGAGA-3′, 21-mer, 2 binding sites,(SEQ ID NO: 19): 5′-TGTGAATTACCGGAAGTG-3′, 18-mer, 2 binding sites, (SEQID NO: 21): 5′-AGTCAGTTCCAGGAACTGACT-3′, 21-mer, 2 binding sites, (SEQID NO: 23): 5′-ATGTGAGTTCCCGGAAGTGAACT-3′, 23-mer, 2 binding sites, (SEQID NO: 25): 5′-ACAGTTCCGGGAACTGTC-3′, 19-mer, 2 binding sites, (SEQ IDNO: 27): 5′-GACAGTTCCGGGAACTGTC-3′, 19-mer, 2 binding sites, (SEQ ID NO:29): 5′-GTGTATTCCGGTAAGTGA-3′, 18-mer, 2 binding sites, (SEQ ID NO: 31):5′-TTATGTGAATTCCTGGAAGTG-3′, 21-mer, 2 binding sites, (SEQ ID NO: 33):5′-CATGTTATGCATATTCCTGTAAGTG-3′, 25-mer, 2 binding sites, (SEQ ID NO:35): 5′-TGTGAATTCCTGTAAGTGAGA-3′, 21-mer, 2 binding sites, (SEQ ID NO:37): 5′-TGCATATTCCTGTAAGTG-3′, 18-mer, 2 binding sites, (SEQ ID NO: 39):5′-ATATTCCTGTAAGTG-3′, 15-mer, 2 binding sites.

The remark “2 binding sites” thereby relates to the sense- andantisense-strand. This listing of the preferred sequences is notlimiting. It is obvious for a person skilled in the art that amultiplicity of sequences can be used as inhibitors for STAT-1 as longas they exhibit the previously denoted conditions of the 11-merconsensus core binding sequence and an affinity to STAT-1.

The affinity of the binding of a nucleic acid sequence to STAT-1 can beassessed by the use of the electrophoretic mobility shift assay (EMSA)(Sambrook et al. (1989), Molecular Cloning. Cold Spring HarborLaboratory Press; Krezesz et al. (1999), FEBS Lett. 453, 191). This testsystem is suited for the quality control of nucleic acids which areintended for the use in the method of the present invention, or for thedetermination of the optimal length of a binding site. It is also suitedfor the identification of other sequences which are bound by STAT-1. Foran EMSA, intended for the isolation of new binding sites, purified orrecombinantly expressed versions of STAT-1 are most suitable which areapplied in several alternating rounds of PCR-amplifications and aselection by EMSA (Thiesen and Bach (1990), Nucleic Acids Res. 18,3203).

Genes known for encompassing STAT-1 binding sites in their promoter orenhancer regions or in the case of genes where there is alreadyfunctional evidence for the importance of STAT-1 in their expression andwhich are therefore presumable targets for the specific squelching bythe method of the present invention, are besides the CD40-, E-selectin-,inducible NO-synthase-, the interleukin-12 (p40)- and the MCP-1-genefurther pro-inflammatory genes, e.g. IFNγ itself, the cytokineinterleukin-6, the adhesion molecules ICAM-1, PECAM-1 (plateletendothelial cell adhesion molecule-1), RANTES (regulated uponactivation, normal T-cell expressed, presumed secreted; solubly secretedby T-lymphocytes) and VCAM-1, the chemokines interleukin-8, IP-10(interferon-inducible protein-10) and Mig (monokine induced bygamma-interferon) as well as the MHC-proteins I and II. Thereby it doesnot matter whether the expression of these genes is regulated by STAT-1directly or indirectly (e.g. via the STAT-1-dependent expression ofIRF-1)

If a decoy-oligonucleotide according to the invention against STAT-1 butnot a respective control-oligonucleotide in human endothelial cells isused, the cytokine-induced expression of CD40 (both in themono-stimulation with IFNγ and in the combination of IFNγ and TNFα) isconsiderably inhibited by more than 50%. This holds true also for theexpression of E-selectin and MCP-1 and interleukin-12 (p40) respectivelyif the stimulation of the cells takes place with IFNγ and TNFα and CD154respectively. According to this an elimination of the STAT-1-activitybrings about a highly significant inhibition of the expression of agroup of pro-inflammatory gene products in human endothelial cells.Insofar one is to figure on a significant reduction of theendothelium-leukocyte-interaction (E-selectin, MCP-1), but also of theinteraction of antigen-presenting cells (e.g. macrophages andB-lymphocytes) with T-lymphocytes (CD40, interleukin-12) in the scope ofinflammatory diseases in the case of this therapeutic approach.Analogously this also holds true for the shown reduction of thecytokine-induced IRF-1-expression in the THP-1-monocytes (and thereby ofthe downstream expression of IRF-1-dependent genes) as well as of thecytokine-induced expression of the mentioned gene products including theinducible NO-synthase in the human smooth muscle cells.

The method of the present invention modulates the transcription of agene or of genes in such a way that the gene or the genes, e.g.E-selectin, is/are not or less expressed. A lessened or suppressedexpression in the scope of the present invention means that the rate oftranscription is decreased in comparison to cells which are not treatedwith a double stranded DNA-oligonucleotide according to the presentinvention. Such a decrease can be determined e.g. bynorthern-blot-analysis (Sambrook et al., 1989) or RT-PCR (Sambrook etal., 1989). Usually such a decrease is at least a 2-fold, in particularat least a 5-fold, particularly at least a 10-fold decrease. The loss ofactivation can be achieved e.g. if STAT-1 acts on a certain gene as atranscriptional activator and therefore the squelching of the activatorleads to the loss of the expression of the target gene.

Furthermore the method of the present invention facilitates the releaseof inhibition of the expression of a gene as far as the expression isblocked by a constitutively active or (after a respective stimulation ofthe cell) by an activated transcription factor. An example for this isthe release of inhibition of the expression of theprepro-endothelin-1-gene in native endothelial cells of the V. jugularisof the rabbit by a cis-element decoy against the transcription factorCCAAT/enhancer binding protein (Lauth et al., J. Mol. Med. (2000), 78,441). By this means the inhibition of the expression of genes can bereleased whose products exert a protective effect e.g. againstinflammatory diseases. So, e.g. the endothelial isoform of theNO-synthase, whose product NO plays a crucial role within thesuppression of the expression of pro-inflammatory adhesion molecules andchemokines in endothelial cells, is down regulated by IFNγ(Rosenkranz-Weiss et al. (1994), J. Clin. Invest. 93, 1875). Acis-element decoy against STAT-1 can reverse this undesired effect byinhibiting the binding of STAT-1 to the according binding site in thepromoter of the endothelial NO-synthase gene.

The cis-element decoy according to the present invention, in a preferredembodiment contains one or more, preferentially 1, 2, 3, 4 or 5,particularly preferred 1 or 2 binding sites being bound by STAT-1specifically. The nucleic acids may be generated synthetically, byenzymatic methods or in cells. The single methods are state of the artand known to a person skilled in the art.

The length of the double stranded DNA-oligonucleotide is at least aslong as a used sequence which specifically binds STAT-1. Usually theused double stranded DNA-oligonucleotide has a length between about11-65, preferentially between about 13-28 and particularly preferredbetween 16-23 bp.

Oligonucleotides are usually rapidly degraded by endo- and exonucleases,especially by DNases and RNases in the cell. Therefore theDNA-oligonucleotides may be modified to stabilise them against thedegradation so that a high concentration of the oligonucleotides ismaintained in the cell during a longer period of time. Usually such astabilisation can be obtained by the introduction of one or moremodified internucleotide bonds.

A successfully stabilised DNA-oligonucleotide does not necessarilycontain a modification at each internucleotide bond. Preferably theinternucleotide bonds at the respective ends of both oligonucleotides ofthe cis-element decoy are modified. Thereby the last six, five, four,three, two or the last or one or more internucleotide bonds within thelast six internucleotide bonds can be modified. Further differentmodifications of the internucleotide bonds can be inserted into thenucleic acid and the thereby emerging double strandedDNA-oligonucleotides can be assayed for the sequence specific binding toSTAT-1 using the routine EMSA-test system. This test system allows thedetermination of the binding constant of the cis-element decoy andtherefore the determination whether the affinity was changed by themodification. Modified cis-element decoys which still show a sufficientbinding can be selected whereby a sufficient binding means at leastabout 50% or at least about 75%, and particularly preferred about 100%of the binding of the unmodified nucleic acid.

Cis-element decoys with modified internucleotide bonds which still showa sufficient binding can be tested if they are more stable in the cellthan the unmodified cis-element decoys. The cells “transfected” with thecis-element decoys according to the invention are assayed for the amountof the still available cis-element decoys at different time points.Thereby preferably a cis-element decoy labelled with a fluorescentdye-stuff (e.g. Texas-red) or a cis-element decoy labelled radioactively(e.g. ³²P) is used with a subsequent digital fluorescence microscopy andautoradiography or scintigraphy respectively. A successfully modifiedcis-element decoy has a half-life in the cell which is higher than thehalf-life of an unmodified cis-element decoy, preferably of at leastabout 48 hours, more preferred of at least about 4 days, most preferredof at least about 7 days.

Suitable modified internucleotide bonds are summarised in Uhlmann andPeyman ((1990) Chem. Rev. 90, 544). Modifiedinternucleotide-phosphate-residues and/or non phosphorus-bridges in anucleic acid which may be used in a method according to the presentinvention contain e.g. methylphosphonate, phosphorothioate,phosphorodithioate, phosphoramidate, phosphate-ester, whereasnon-phosphorus internucleotide-analogues contain e.g. siloxane-bridges,carbonate-bridges, carboxymethylester-bridges, acetamidate-bridgesand/or thioether-bridges. In the case of the use ofphosphorothioate-modified internucleotide bonds they preferably shouldnot lie between the bases cytosine and guanine since that may lead to anactivation of the target cells of the cis-element decoy.

A further embodiment of the invention is the stabilisation of nucleicacids by the insertion of structural characteristics into the nucleicacids which increase the half-life of the nucleic acid. Such structurescontaining hairpin- and bell-shaped DNA, are disclosed in U.S. Pat. No.5,683,985. At the same time, modified internucleotide-phosphate-residuesand/or non-phosphorus-bridges can be introduced together with thementioned structures. The thereby resulting nucleic acids can be assayedin the above described test system for binding and stability.

The core binding sequence may not only be present in a cis-element decoybut also in a vector. In a preferred embodiment the vector is a plasmidvector and in particular a plasmid vector which is able to replicateautosomally thereby increasing the stability of the introduced doublestranded nucleic acid.

A cis-element decoy of the present invention is quickly taken up intothe cell. A sufficient uptake is characterised by the modulation of theexpression of one or more genes which are subject to a control bySTAT-1. The cis-element decoy of the present invention preferablymodulates the transcription of a gene or of genes after about 4 hoursafter contacting the cell, more preferred after about 2 hours, afterabout 1 hour, after about 30 minutes and most preferred after about 10minutes. A typical mixture being used in such an experiment contains 10μmol/l cis-element decoy.

Furthermore the present invention relates to a method for the modulationof the transcription of at least one gene in cells taking part in theinflammatory events, particularly in endothelial cells, epithelialcells, leukocytes, smooth muscle cells, keratinocytes or fibroblasts,comprising the step of contacting the mentioned cells with a mixturecontaining one or more double stranded nucleic acids according to theinvention which are able to bind sequence-specifically to thetranscription factor STAT-1. A preferred method is e.g. the ex vivotreatment of a donation of bone marrow containing T-lymphocytes prior tothe introduction into the recipient's body.

Furthermore the cis-element decoys according to the invention can beadministered to the patients in a composition or be used in the methodaccording to the invention. The composition (in the following calledmixture) containing the cis-element decoys according to the invention isbrought into contact with the target cells (e.g. endothelial cells,epithelial cells, leukocytes, smooth muscle cells, keratinocytes orfibroblasts). The aim of this contacting is the transfer of thecis-element decoys, which bind STAT-1, into the target cell (i.e. thecell which expresses pro-inflammatory gene products in aSTAT-1-dependent manner). Therefore modifications of nucleic acidsand/or additives or auxiliary substances known to be improving thepenetration of the membrane can be used in the scope of the presentinvention (Uhlmann and Peyman (1990), Chem. Rev. 90, 544).

In a preferred embodiment the mixture according to the inventioncontains only nucleic acid and buffer. A suitable concentration of thecis-element decoys resides in the range of at least 0.1 to 100 μM,preferably at 10 μM, thereby one or more suitable buffers being added.One example of such buffers is Ringer's-solution containing 145 mmol/lNa⁺, 5 mmol/l K⁺, 156 mmol/l Cl⁻, 2 mmol/l Ca²⁺, 1 mmol/l Mg²⁺, 10mmol/l HEPES, 10 mmol/l D-glucose, pH 7.4.

In a further embodiment of the invention the mixture additionallycontains at least one additive and/or auxiliary substance. Additivesand/or auxiliary substances like lipid, cationic lipid, polymers,liposomes, nanoparticles, nucleic acid-aptameres, peptides and proteinswhich are DNA-bound or synthetic peptide-DNA-molecules are intended inorder to (i) increase e.g. the introduction of nucleic acids into thecell, in order to (ii) target the mixture only to a sub-group of cells,in order to (iii) inhibit the degradation of the nucleic acid in thecell, in order to (iv) facilitate the storage of the mixture of thenucleic acids prior to their use. Examples for peptides and proteins orsynthetic peptide-DNA-molecules are e.g. antibodies, fragments ofantibodies, ligands, adhesion molecules which may all of them bemodified or unmodified.

Additives that stabilise the cis-element decoys inside the cell are e.g.nucleic acid-condensing substances like cationic polymers, poly-L-lysineor polyethyleneimine.

The mixture which is used in the method of the present invention ispreferentially applied locally by injection, catheter, suppository,aerosols (nasal and oral spray respectively, inhalation), trocars,projectiles, pluronic gels, polymers with a sustained release ofmedicaments, or any other device facilitating the local access. The exvivo use of the mixture, used in the method of the present invention,allows a local access, too.

But the inhibition of the STAT-1 activity can not only be inhibited onprotein level in the previously described methods but can beaccomplished already before or during the translation of thetranscription factor protein. Therefore it is a further aspect of thepresent invention to provide an inhibitor of the STAT-1-proteinexpression as a therapeutic agent. This inhibitor is preferentially asingle stranded nucleic acid molecule, a so calledantisense-oligonucleotide. Antisense-oligonucleotides can inhibit thesynthesis of a target gene on three different levels, during thetranscription (prevention of the hnRNA-synthesis), during the processing(splicing) of the hnRNA resulting in the mRNA and during the translationof the mRNA into protein at the ribosomes. The method for the inhibitionof the expression of genes by means of antisense-oligonucleotides isstate of the art and well-known to persons skilled in the art. A singlestranded nucleic acid molecule with any sequence can be used as anantisense-oligonucleotide as long as the antisense-oligonucleotide isable to inhibit the STAT-1-protein expression. Preferentially theantisense-oligonucleotide used in the method according to the presentinvention against STAT-1 has the sequence 5′-TACCACTGAGACATCCTGCCAC-3′(SEQ ID NO:41) and bridges the start codon. Further preferred sequencesfor antisense-oligonucleotides are 5′-AACATCATTGGCACGCAG-3′ (SEQ ID NO:42) and 5′-GTGAACCTGCTCCAG-3′ (SEQ ID NO: 43). Theantisense-oligonucleotide can be a single stranded DNA-molecule, anRNA-molecule or a DNA/RNA-hybrid-molecule. The antisense-oligonucleotidecan furthermore exhibit one or more modified internucleotide bonds, e.g.as described previously for the cis-element decoy. In the case of anantisense-oligonucleotide which is stabilised by phosphothioate-modifiedinternucleotide bonds it is to be considered in particular that betweenthe bases cytosine and guanine no phosphorothioate-modifiedinternucleotide bonds are inserted because this leads to an IFNγ-similaractivation of—in particular—immune-competent cells (e.g. endothelialcells) and would therefore, at least partly, foil the desiredtherapeutic effect.

The antisense-oligonucleotides according to the invention can also beused in a composition and be administered to the patients. Thecomposition can be made up of stabilising additives or auxiliarysubstances facilitating e.g. the introduction of theantisense-oligonucleotides into the cell, targeting the composition toonly one subgroup of cells, preventing e.g. the degradation of theantisense-oligonucleotides inside the cell, or facilitating e.g. thestorage of the antisense-oligonucleotide prior to use.

The antisense-oligonucleotide can not only be administered as a singlestranded nucleic acid molecule but can also be present in a vector. In apreferred embodiment the vector is a plasmid vector and in particular aplasmid vector which is able to replicate autosomally thereby increasingthe stability of the introduced single stranded nucleic acid.

A further aspect of the present invention is therefore anantisense-expression vector being expressed inside the target cells bythem after transfection and specifically inhibiting the STAT-1expression. Thereby any available eukaryotic expression vectorsaccording to the state of the art may be concerned. Preferably thepCI-plasmid of the company Promega (Catalogue No. E1731, GenBankAccession Number U47119) is concerned, in which e.g. a 2350 bpcomprising segment of the STAT-1 gene (−121 to +2229, GenBank AccessionNumber XM010893) has been cloned in the opposite direction (3′→5′). Thissegment of the STAT-1 gene is flanked by two EcoRI restriction sites andcontains a XhoI restriction site. Its expression is subjected to thecontrol of the CMV-promoter. The entire plasmid (termed pCI/Stat1 AS)comprises 6365 bp.

As described in Fire (1999), Trends Genet. 15, 358, and Elbashir et al.(2001), Nature 411, 494, furthermore the dsRNA-interference is apreferred method for the inhibition of the STAT-1 activity on the levelof the translation of the mRNA into the transcription factor protein. Inthe case of this method an RNA-double strand comprising exactly 21nucleotides—whose sequence is identical with a segment of the codingmRNA of the target protein (STAT-1)—is introduced into the cell.Subsequently a complex of proteins not being known in detail by now isformed which cleaves specifically the target mRNA thereby preventing itstranslation. Longer RNA-double strands cannot be used because theyelicit a response in the target cells which is comparable to thereaction of the cells to a (viral) infection and would insofar foil thedesired therapeutic effect. Usually thedsRNA-interference-oligonucleotide exhibits one or more internucleotidebonds, e.g. as described previously for the cis-element decoy.

The mixture containing the dsRNA-interference-oligonucleotides accordingto the invention is brought into contact with the target cells (e.g.endothelial cells, epithelial cells, leukocytes, smooth muscle cells,keratinocytes or fibroblasts). Thereby usually additives or auxiliarysubstances known to be improving the penetration of the membrane areused (Uhlmann and Peyman (1990), Chem. Rev. 90, 544).

The following figures and examples serve only for illustration and donot limit the scope of the invention in any respect.

1. Cell Culture

Human endothelial cells were isolated from the veins of the umbilicalcord by treatment with 1.6 U/ml dispase in HEPES-modifiedtyrode-solution for 30 min. at 37° C. and were cultivated ongelatine-coated 6-well-tissue culture dishes (2 mg/ml gelatine in 0.1 MHCl for 30 min. at room temperature) in 1.5 ml M199 medium (Gibco LifeTechnologies, Karlsruhe, Germany), containing 20% foetal calf serum, 50U/ml penicillin, 50 μg/ml streptomycin, 10 U/ml nystatin, 5 mM HEPES and5 mM TES, 1 μg/ml heparin and 40 μg/ml endothelial growth factor. Theywere identified by their typical paving stone morphology, positiveimmune staining for the von Willebrandt-factor (vWF) and by fluorimetricdetection (FACS) of PECAM-1 (CD31) as well as negative immune stainingfor smooth-muscular α-actin (Krzesz et al. (1999), FEBS Lett. 453, 191).

The human monocyte-cell line TBP-1 (ATCC TIB 202) was cultivated in RPMI1640 medium (Life Technologies) containing 10% foetal calf serum, 50U/ml penicillin, 50 μg/ml streptomycin and 10 U/ml nystatin. The humansmooth muscle cells were isolated from dissected thymus veins by meansof the explant-technology (Krzesz et al. (1999), FEBS Lett. 453, 191)and cultivated on gelatine-coated 6-well-tissue culture dishes (seeabove) in 1.5 ml Dulbecco's modified eagle medium, containing 15% foetalcalf serum, 50 U/ml penicillin, 50 μg/ml streptomycin and 10 U/mlnystatin. They were identified by positive immune staining for smoothmuscular α-actin.

2. RT-PCR-Analysis

The endothelial total-RNA was isolated with the Qiagen RNeasy kit(Qiagen, Hilden, Germany) followed by a cDNA-synthesis with a maximum of3 μg RNA and 200 U Superscript™ II reverse transcriptase (LifeTechnologies) in a total volume of 20 μl according to the manufacturersprotocol. For the adjustment of the cDNA-loading 5 μl (about 75 ng cDNA)of the resulting cDNA-solution and the primer pair (Gibco) for theelongation factor 1 (EF-1)-PCR with 1 U Taq DNA polymerase (Gibco) wereused in a total volume of 50 μl. EF-1 served as an internal standard forthe PCR. The PCR-products were separated on 1.5% agarose-gels containing0.1% ethidium bromide and the intensity of the bands was determineddensitometrically with a CCD-camera system and the One-Dscan gelanalysis-software of Scanalytics (Billerica, Mass., USA) in order toadjust the volume of the cDNA in the following PCR-analysis.

All PCR-reactions were performed separately for each primer pair in aHybaid OmnE Thermocycler (AWG; Heidelberg, Germany). The individualPCR-conditions for the cDNA of human endothelial cells from theumbilical cord were as follows: CD40 (product size 381 bp, 25 cycles,annealing temperature 60° C., (forward primer)5′-CAGAGTTCACTGAAACGGAATGCC-3′ (SEQ ID NO: 44), (reverse primer)5′-TGCCTGCCTGTTGCACAACC-3′ (SEQ ID NO: 45)); E-selectin (product size304 bp, 33 cycles, annealing temperature 60° C., (forward primer)5′-AGCAAGGCATGATGTTAACC-3′ (SEQ ID NO: 46), (reverse primer)5′-GCATTCCTCTCTTCCAGAGC-3′ (SEQ ID NO: 47)); EF-1 (product size 220 bp,22 cycles, annealing temperature 55° C., (forward primer)5′-TCTTAATCAGTGGTGGAAG-3′ (SEQ ID NO: 48), (reverse primer)5′-TTTGGTCAAGTTGTTTCC-3′ (SEQ ID NO: 49)); IL-12p40 (product size 281bp, 30 cycles, annealing temperature 62° C., (forward primer)5′-GTACTCCACATTCCTACTTCTC-3′ (SEQ ID NO: 50), (reverse primer)5′-TTTGGGTCTATTCCGTTGTGTC-3′ (SEQ ID NO: 51)); rp132 (product size 368bp, 20 cycles, annealing temperature 60° C., (forward primer)5′-GTTCATCCGGCACCAGTCAG-3′ (SEQ ID NO: 52), (reverse primer)5′-ACGTGCACATGAGCTGCCTAC-3′ (SEQ ID NO: 53); MCP-1 (product size 330 bp,22 cycles, annealing temperature 63° C., (forward primer)5′-GCGGATCCCCTCCAGCATGAAAGTCTCT-3′ (SEQ ID NO: 54), (reverse primer)5′-ACGAATTCTTCTTGGGTTGTGGAGTGAG-3′ (SEQ ID NO: 55).

3. Electrophoretic Mobility Shift Assay (EMSA)

The nuclear extracts and [³²P]-labelled double strandedconsensus-oligonucleotides (Santa Cruz Biotechnologie, Heidelberg,Germany), non-denaturing polyacrylamide-gel electrophoresis,autoradiography and supershift-analysis were performed as described inKrzesz et al. (1999), FEBS Lett. 453, 191. Thereby a double strandedDNA-oligonucleotide was used having the following single strandedsequence (the core binding sequence is underlined): SIE,5′-GTGCATTTCCCGTAAATCTTGTC-3′ (SEQ ID NO: 56). For the analysis of theextrusion of endogenous STAT-1 in nuclear extracts ofcytokine-stimulated THP-1-cells (pre-monocytous human cell line) by thevarious cis-element decoys, a ratio of 30:1 (STAT-1 cis-element decoy:[³²P]-labelled SEE oligonucleotide (11 fmol)) was chosen in theEMSA-binding approach.

4. Decoy-Oligonucleotide-Technique

Double stranded decoy-oligonucleotides were generated with thecomplementary single stranded phosphorothioate-linked oligonucleotides(Eurogentec, Köln, Germany) as described in Krzesz et al. (1999), FEBSLett. 453, 191. The cultivated human endothelial cells werepre-incubated at a concentration of 10 μM of the respectivedecoy-oligonucleotide for 4 hours. These were the conditions which werealready previously optimised, based on the EMSA and RT-PCR-analysis.After this, the decoy-oligonucleotide containing medium was usuallyreplaced by fresh medium. The single stranded sequences of theoligonucleotide were as follows (the underlined letters indicatephosphorothioate-linked bases, each of them in 5′-3′ direction):

(SEQ ID NO: 57) GATA-2, CACTTGATAACAGAAAGTGATAACTCT (SEQ ID NO: 58)NF-κB, AGTTGAGGGGACTTTCCCAGGC; (SEQ ID NO: 33) STAT-1,CATGTTATGCATATTCCTGTAAGTG; (SEQ ID NO: 59) STAT-1-19mut,GACAGTGCAGTGAACTGTC; (SEQ ID NO: 60) STAT-1-25mut,CATGTTATGCAGACCGTAGTAAGTG.

5. Antisense-Oligonucleotide (ODN)-Technique

For an antisense-approach 100 ml OPTI-MEM®I culture medium was spikedwith 15 μl lipofectin (Gibco Life Technologie, Karlsruhe, Germany) andincubated at room temperature (RT) for 45 minutes (solution A).Subsequently the antisense-ODN (Eurogentec, Köln, Germany) was added toa final concentration of 0.5 μM in 100 μL OPTI-MEM®I culture medium(solution B). After pooling the solutions A and B a further incubationfor 15 minutes (RT) followed. At the start of the experiments 0.8 ml ofthe conventional cell culture medium of the culture of the endothelialcells (without heparin and endothelial growth factor) were added to anEppendorf-tube containing the lipofectin-antisense-ODN-complexes and thecell culture medium of the culture of endothelial cells was replaced bythe antisense-lipofectin-medium. The antisense-lipofectin-medium wasremoved after 4 hours and replaced by a fresh cell culture medium (withheparin and endothelial growth factor). The sequence of theSTAT-1-antisense-ODN was 5′-T*A*CCA*C*T*G*A*G*A*C*A*T*CC*T*GCC*A*C-3′ (*phosphorothioate-modified base; SEQ ID NO: 41).

6. Western Blot-Analysis

The human endothelial cells from the umbilical cord and smooth musclecells from the thymus vein were cracked by subsequent freezing in liquidnitrogen and thawing at 37° C. (thermoblock, Kleinfelden, Germany) forfive times. Protein extracts were prepared as described in Hecker et al.(1994), Biochem J. 299, 247. 20-30 μg protein were separated by means ofa 10% polyacrylamide gel electrophoresis under denaturing conditions inthe presence of SDS following the standard protocol and transferred to aBioTrace™ polyvinylidene fluoride transfer membrane (Pall Corporation,Roβdorf, Germany). The following primary antibodies were used for theimmunological protein detection: CD40 protein (polyclonal, 1:2000dilution, Research Diagnostics Inc., Flanders N.J., USA), STAT-1 protein(monoclonal, 1:5000 dilution, BD Transduction Laboratories, Heidelberg,Germany), IRF-1 protein (polyclonal, 1:2000 dilution, Santa CruzBiotechnology, Heidelberg, Germany), iNOS protein (polyclonal, 1:3000dilution, BD Transduction Laboratories, Heidelberg, Germany). Theprotein bands were detected after the addition of a peroxidase-linkedanti-rabbit-IgG and—in the case of the use of the monoclonal primaryantibody—by a respective anti-mouse-IgG (1:3000, Sigma, Deisenhofen,Germany) respectively by means of the chemiluminescence method(SuperSignal Chemiluminescent Substrate; Pierce Chemical, Rockford,Ill., USA) and a subsequent autoradiography (Hyperfilm™ MP, AmershamPharmacia, Biotech, Buckinghamshireiii, England). The loading and thetransfer of equal protein amounts was shown after “stripping” of thetransfer membrane (5 minutes 0.2 N NaOH, followed by washing with H₂Ofor 3×10 minutes) by the detection of equal protein bands of β-actinwith a monoclonal primary antibody and a peroxidase-linked anti-mouseIgG (both from Sigma-Aldrich, 1:3000 dilution).

7. Statistical Analysis

If not indicated differently all data in the figures and text aredenoted as a mean value SEM of n experiments. The statistical evaluationwas performed with the students t-test for unpaired data with ap-value<0.05 which was considered as statistically significant.

8. Detection of the Effect of Decoy-Oligonucleotides by Experimentationon Animals 8.1 Mouse

For the detection of the efficiency of the decoy-oligonucleotide-basedtherapeutic approach developed in the present application an animalexperiment related proof-of-concept-study in the mouse with 8-10 animalsper group was performed for the indication of an antigen-inducedarthritis (for the model see Henzgen et al., Exp. Toxicol. Pathol.(1996), 48, 255). A single application of 0.25 nmol of theSTAT-1-decoy-oligonucleotide (SEQ ID NO: 33) directly into the joint(intra-articular injection) reduced high significantly theantigen-induced swelling of the joint (by 35%), the intensity of theinflammatory response (by 70%), the articular destruction (by 80%), thetotal arthritis-score (by 70%) and the concentration of pro-inflammatorycytokines in the serum (e.g. interleukin-6 by 80%) during a period of3-14 days. In contrast, the respective control-oligonucleotide had notherapeutic effect.

Furthermore it was noteworthy in this study that the contact dermatitis(type-IV-reaction) which was elicited in the skin 14 days after theinduction of the arthritis—thereby the antigen is once more injectedsubcutaneously into the animals—was also high significantly inhibited(by 35%) in the decoy-oligonucleotide treated mice.

8.2 Guinea Pig

After allergisation of the guinea pigs for two times (Hartley, male, 350g body weight) during a period of 7 days (on day 1 in one ear, on day 2in the other ear with 50 μl of a 10% DNCB-solution in 50% acetone/50%olive oil each; on day 7 a boost in the skin of the neck with 15 μl of a2% DNCB-solution in 95% acetone/5% olive oil) the contact dermatitis iselicited by a re-application of 2,4-dinitrochorobenzol (DNCB; 10 μl of a0.5% solution of DNCB in 95% acetone/5% olive oil) on day 13 on one andmore areas being of about 1 cm² in size respectively on the shaved backsof the animals and assessed macroscopically and histologically 24 hourslater. The contact dermatitis induced in such a way is histologically(Giemsa-staining) characterised by a pronounced formation of oedema andspongiosis in the area of the epidermis, an increase of apoptotic cellsas well as massive infiltration by leukocytes (FIG. 8). The intradermalapplication of a STAT-1-decoy-oligonucleotide (SEQ ID NO: 19) but not ofa mutated control-oligonucleotide (5′-TGTGGACCGTAGGAAGTG-3′, SEQ ID NO:61) 1 hour before the final DNCB-exposition led to a clear reduction ofthe mentioned histological parameters, i.e. in total to a significantattenuation of the inflammatory response.

1. A method for the prevention or therapy of a disease state associatedwith STAT-1 activity comprising administering to a subject in needthereof a double-stranded DNA-oligonucleotide, a single strandedantisense-oligonucleotide, an antisense-expression vector or adouble-stranded RNA-interference-oligonucleotide that inhibits STAT-1activity.
 2. The method according to claim 1, wherein the disease stateis cardio-vascular complications like restenosis after percutaneousangioplasty or stenosis of venous bypasses, the graft versus hostreaction, the ischemia/refusion-related damage in the context ofsurgical interventions and organ transplantation respectively,immunological hypersensitivity reactions, in particular the allergicrhinitis, the drug and food allergies, in particular urticaria andceliac disease (sprue), contact eczema and the immune complex diseases,in particular alveolitis, arthritis, glomerulonephritis and allergicvasculitis, inflammatory chondro- and osteopathies, in particulararthrosis, gout, ostitis and osteomyelitis, polyneuritis as well asacute and subacute respectively, infection contingent and in particularpost-infectious inflammatory diseases, in particular bronchitis,endocarditis, hepatitis, myocarditis, nephritis, pericarditis,peritonitis or pancreatitis, including the septic shock.
 3. The methodof claim 1, wherein said double-stranded DNA-oligonucleotide isadministered.
 4. The method of claim 1, wherein said single strandedantisense-oligonucleotide is administered.
 5. The method of claim 1,wherein said antisense-expression vector is administered.
 6. The methodof claim 5, wherein the vector is a plasmid vector.
 7. The method ofclaim 1, wherein said double-stranded RNA-interference-oligonucleotideis administered.
 8. The method of claim 1, wherein said double-strandedDNA-oligonucleotide, said single stranded antisense-oligonucleotide,said antisense-expression vector or said double-strandedRNA-interference-oligonucleotide is administered by injection, catheter,suppository, aerosol, trocar, projectile, pluronic gel or polymer. 9.The method of claim 1, further comprising ex vivo administration of saiddouble-stranded DNA-oligonucleotide, said single strandedantisense-oligonucleotide, said antisense-expression vector or saiddouble-stranded RNA-interference-oligonucleotide.
 10. The method ofclaim 1, wherein administering brings said double-strandedDNA-oligonucleotide, said a single stranded antisense-oligonucleotide,said antisense-expression vector or said double-strandedRNA-interference-oligonucleotide into contact with an endothelial cell,an epithelial cell, a leukocyte, a smooth muscle cell, a keratinocyte ora fibroblast.